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Objective: During hypoinsulinemia, when cardiac glucose utilization is impaired, the heart rapidly adapts to using more fatty acids. One means by which this is achieved is through lipoprotein lipase (LPL). We determined the mechanisms by which the heart regulates LPL after acute hypoinsulinemia.Research Design and Methods: We used two different doses of streptozocin (55 [D-55] and 100 [D-100] mg/kg) to induce moderate and severe hypoinsulinemia, respectively, in rats. Isolated cardiomyocytes were also used for transfection or silencing of protein kinase D (PKD) and caspase-3.Results: There was substantial increase in LPL in D-55 hearts, an effect that was absent in severely hypoinsulinemic D-100 animals. Measurement of PKD, a key element involved in increasing LPL, revealed that only D-100 hearts showed an increase in proteolysis of PKD, an effect that required activation of caspase-3 together with loss of 14-3-3zeta, a binding protein that protects enzymes against degradation. In vitro, phosphomimetic PKD colocalized with LPL in the trans-golgi. PKD, when mutated to prevent its cleavage by caspase-3 and silencing of caspase-3, was able to increase LPL activity. Using a caspase inhibitor (Z-DEVD) in D-100 animals, we effectively lowered caspase-3 activity, prevented PKD cleavage, and increased LPL vesicle formation and translocation to the vascular lumen. This increase in cardiac luminal LPL was associated with a striking accumulation of cardiac triglyceride in Z-DEVD-treated D-100 rats. CONCLUSIONS After severe hypoinsulinemia, activation of caspase-3 can restrict LPL translocation to the vascular lumen. When caspase-3 is inhibited, this compensatory response is lost, leading to lipid accumulation in the heart. [ABSTRACT FROM AUTHOR]

Conventional models view β 1 -adrenergic receptors (β 1 ARs) as full-length proteins that activate signaling pathways that influence contractile function and ventricular remodeling - and are susceptible to agonist-dependent desensitization. This perspective summarizes recent studies from my laboratory showing that post-translational processing of the β 1 -adrenergic receptor N-terminus results in the accumulation of both full-length and N-terminally truncated forms of the β 1 AR that differ in their signaling properties. We also implicate oxidative stress and β 1 AR cleavage by elastase as two novel mechanisms that would (in the setting of cardiac injury or inflammation) lead to altered or decreased β 1 AR responsiveness., Competing Interests: The authors declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Steinberg.)

The decrease in β 1 -adrenergic receptor responsiveness in heart failure is attributed conventionally to agonist-dependent desensitization. We identify elastase-dependent β 1 -adrenergic receptor cleavage as a novel proteolytic mechanism that disrupts β 1 -adrenergic receptor responsiveness in the setting of tissue injury or inflammation., Competing Interests: This work was funded by NHLBI grant RO1-HL138468. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes. NEW & NOTEWORTHY PKCα is an important signaling molecule extensively studied in many cellular processes. However, no isoforms have been reported for PKCα except one prototypic isoform. Alternative mRNA splicing of Prkca gene was detected for the first time in rodent and human cardiac tissue, which can produce a previously unknown PKCα-novel exon (NE) isoform. The biochemistry and molecular effects of PKCα-NE are markedly different from PKCα wild type, suggesting potential functional diversity of PKCα signaling in muscle.

Abstract: β 1 -adrenergic receptors (β 1 ARs) are the principle mediators of catecholamine actions in cardiomyocytes. β 1 ARs rapidly adjust cardiac output and provide short-term hemodynamic support for the failing heart by activating a Gs-adenylyl cyclase pathway that increases 3'-5'-cyclic adenosine monophosphate and leads to the activation of protein kinase A and the phosphorylation of substrates involved in excitation-contraction coupling. However, chronic persistent β 1 AR activation in the setting of heart failure leads to a spectrum of maladaptive changes that contribute to the evolution of heart failure. The molecular basis for β 1 AR-driven maladaptive responses remains uncertain because chronic persistent β 1 AR activation has been linked to the activation of both proapoptotic and antiapoptotic signaling pathways. Of note, studies to date have been predicated on the assumption that β 1 ARs signal exclusively as full-length receptor proteins. Our recent studies show that β 1 ARs are detected as both full-length and N-terminally truncated species in cardiomyocytes, that N-terminal cleavage is regulated by O-glycan modifications at specific sites on the β 1 AR N-terminus, and that N-terminally truncated β 1 ARs remain signaling competent, but their signaling properties differ from those of the full-length β 1 AR. The N-terminally truncated form of the β 1 AR constitutively activates the protein kinase B signaling pathway and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. These studies identify a novel signaling paradigm for the β 1 AR, implicating the N-terminus as a heretofore-unrecognized structural determinant of β 1 AR responsiveness that could be pharmacologically targeted for therapeutic advantage., Competing Interests: The author reports no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Competing Interests: The authors report no conflicts of interest.

β 1 -Adrenergic receptors (β 1 ARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that β 1 ARs accumulate as both full-length and NH 2 -terminally truncated species in cells, that maturational processing of full-length β 1 ARs to an NH 2 -terminally truncated form is attributable to O -glycan-regulated proteolytic cleavage of the β 1 AR NH 2 -terminus at R 31  ↓ L 32 by ADAM17, and that NH 2 -terminally truncated β 1 ARs remain signaling competent but they acquire a distinct signaling phenotype. NH 2 -terminally truncated β 1 ARs differ from full-length β 1 ARs in their signaling bias to cAMP/PKA versus ERK pathways and only the NH 2 -terminally truncated form of the β 1 AR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. Since the R 31  ↓ L 32 sequence conforms to a trypsin consensus cleavage site, we used immunoblotting methods to test the hypothesis that β 1 ARs are also cleaved at R 31  ↓ L 32 by trypsin (an enzyme typically used to isolate cardiomyocytes from the intact ventricle). We show that full-length β 1 ARs are cleaved by trypsin and that trypsin cleaves the full-length β 1 AR NH 2 -terminus specifically at R 31  ↓ L 32 in CHO-Pro5 cells. Trypsin also cleaves β 1 ARs in cardiomyocytes, but at a second site that results in the formation of ∼40-kDa NH 2 -terminal and ∼30-kDa COOH-terminal fragments. The observation that cardiomyocyte β 1 ARs are cleaved by trypsin (a mechanism that constitutes a heretofore-unrecognized mechanism that would influence β 1 AR-signaling responses) suggests that studies that use standard trypsin-based procedures to isolate adult cardiomyocytes from the intact ventricle should be interpreted with caution. NEW & NOTEWORTHY Current concepts regarding the molecular basis for β 1 AR responses derive from literature predicated on the assumption that β 1 ARs signal exclusively as full-length receptor proteins. However, we recently showed that β 1 ARs accumulate as both full-length and NH 2 -terminally truncated forms. This manuscript provides novel evidence that β 1 -adrenergic receptors can be cleaved by trypsin and that cell surface β 1 AR cleavage constitutes a heretofore unrecognized mechanism to alter catecholamine-dependent signaling responses.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that play key roles in a wide range of biological functions that contribute to the evolution of cardiac hypertrophy and heart failure. PKD1 (the founding member of this enzyme family) has been implicated in the phosphorylation of substrates that regulate cardiac hypertrophy, contraction, and susceptibility to ischemia/reperfusion injury, and de novo PRKD1 (protein kinase D1 gene) mutations have been identified in patients with syndromic congenital heart disease. However, cardiomyocytes coexpress all three PKDs. Although stimulus-specific activation patterns for PKD1, PKD2, and PKD3 have been identified in cardiomyocytes, progress toward identifying PKD isoform-specific functions in the heart have been hampered by significant gaps in our understanding of the molecular mechanisms that regulate PKD activity. This review incorporates recent conceptual breakthroughs in our understanding of various alternative mechanisms for PKD activation, with an emphasis on recent evidence that PKDs activate certain effector responses as dimers, to consider the role of PKD isoforms in signaling pathways that drive cardiac hypertrophy and ischemia/reperfusion injury. The focus is on whether the recently identified activation mechanisms that enhance the signaling repertoire of PKD family enzymes provide novel therapeutic strategies to target PKD enzymes and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling. SIGNIFICANCE STATEMENT: PKD isoforms regulate a large number of fundamental biological processes, but the understanding of the biological actions of individual PKDs (based upon studies using adenoviral overexpression or gene-silencing methods) remains incomplete. This review focuses on dimerization, a recently identified mechanism for PKD activation, and the notion that this mechanism provides a strategy to develop novel PKD-targeted pharmaceuticals that restrict proliferation, invasion, or angiogenesis in cancer and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: We describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease., Methods: We performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins., Results: We identified two different de novo mutations in protein kinase D1 ( PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 ( PKD1 , HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation., Conclusion: The present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

β 1 -adrenergic receptors (β 1 ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the β 1 AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the β 1 AR N-terminus carries O-glycan modifications at Ser 37 /Ser 41 , that O-glycosylation prevents β 1 AR N-terminal cleavage, and that N-terminal truncation influences β 1 AR signaling to downstream effectors. However, the site(s) and mechanism for β 1 AR N-terminal cleavage in cells was not identified. This study shows that β 1 ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated β 1 AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R 31 ↓L 32 . We identify Ser 41 as the major O-glycosylation site on the β 1 AR N-terminus and show that an O-glycan modification at Ser 41 prevents ADAM17-dependent cleavage of the β 1 -AR N-terminus at S 41 ↓L 42 , a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates β 1 -AR N-terminal cleavage at R 31 ↓L 32 ). We previously reported that oxidative stress leads to a decrease in β 1 AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte β 1 ARs is via a mechanism involving N-terminal truncation at R 31 ↓L 32 by ADAM17. In keeping with the previous observation that N-terminally truncated β 1 ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant β 1 AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the β 1 AR N-terminus as a structural determinant of β 1 AR responses that can be targeted for therapeutic advantage., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild type up to 36 h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Protein kinases are a superfamily of enzymes that control a wide range of cellular functions. These enzymes share a highly conserved catalytic core that folds into a similar bilobar three-dimensional structure. One highly conserved region in the protein kinase core is the glycine-rich loop (or G-loop), a highly flexible loop that is characterized by a consensus GxGxxG sequence. The G-loop points toward the catalytic cleft and functions to bind and position ATP for phosphotransfer. Of note, in many protein kinases, the second and third glycine residues in the G-loop triad flank residues that can be targets for phosphorylation (Ser, Thr, or Tyr) or other post-translational modifications (ubiquitination, acetylation, O-GlcNAcylation, oxidation). There is considerable evidence that cyclin-dependent kinases are held inactive through inhibitory phosphorylation of the conserved Thr/Tyr residues in this position of the G-loop and that dephosphorylation by cellular phosphatases is required for CDK activation and progression through the cell cycle. This review summarizes literature that identifies residues in or adjacent to the G-loop in other protein kinases that are targets for functionally important post-translational modifications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The mechanism that leads to a decrease in β 1 -adrenergic receptor (β 1 AR) expression in the failing heart remains uncertain. This study shows that cardiomyocyte β 1 AR expression and isoproterenol responsiveness decrease in response to oxidative stress. Studies of mechanisms show that the redox-dependent decrease in β 1 AR expression is uniquely prevented by carvedilol and not other βAR ligands. Carvedilol also promotes the accumulation of N-terminally truncated β 1 ARs that confer protection against doxorubicin-induced apoptosis in association with activation of protein kinase B. The redox-induced molecular controls for cardiomyocyte β 1 ARs and pharmacologic properties of carvedilol identified in this study have important clinical and therapeutic implications.

Protein kinase C-δ (PKCδ) is an allosterically activated enzyme that acts much like other PKC isoforms to transduce growth factor-dependent signaling responses. However, PKCδ is unique in that activation loop (Thr 507 ) phosphorylation is not required for catalytic activity. Since PKCδ can be proteolytically cleaved by caspase-3 during apoptosis, the prevailing assumption has been that the kinase domain fragment (δKD) freed from autoinhibitory constraints imposed by the regulatory domain is catalytically competent and that Thr 507 phosphorylation is not required for δKD activity. This study provides a counternarrative showing that δKD activity is regulated through Thr 507 phosphorylation. We show that Thr 507 -phosphorylated δKD is catalytically active and not phosphorylated at Ser 359 in its ATP-positioning G-loop. In contrast, a δKD fragment that is not phosphorylated at Thr 507 (which accumulates in doxorubicin-treated cardiomyocytes) displays decreased C-terminal tail priming-site phosphorylation, increased G-loop Ser 359 phosphorylation, and defective kinase activity. δKD is not a substrate for Src, but Src phosphorylates δKD-T507A at Tyr 334 (in the newly exposed δKD N terminus), and this (or an S359A substitution) rescues δKD-T507A catalytic activity. These results expose a unique role for δKD-Thr 507 phosphorylation (that does not apply to full-length PKCδ) in structurally organizing diverse elements within the enzyme that critically regulate catalytic activity., (Copyright © 2017 American Society for Microbiology.)

β 1 -adrenergic receptors (β 1 ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of the β 1 AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the β 1 AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies β 1 AR N-terminal O-glycosylation at Ser 37 /Ser 41 as a mechanism that prevents β 1 AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated β 1 ARs and N-terminally truncated glycosylation-defective β 1 ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes β 1 ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of β 1 AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that are co-expressed in the heart and have important roles in many biological responses. PKD1 is activated by pro-hypertrophic stimuli and has been implicated in adverse cardiac remodeling. Efforts to define the cardiac actions of PKD2 and PKD3 have been less successful at least in part because conventional methods provide a general screen for PKD activation but are poorly suited to resolve activation patterns for PKD2 or PKD3. This study uses Phos-tag SDS-PAGE, a method that exaggerates phosphorylation-dependent mobility shifts, to overcome this technical limitation. Phos-tag SDS-PAGE resolves PKD1 as distinct molecular species (indicative of pools of enzyme with distinct phosphorylation profiles) in unstimulated cardiac fibroblasts and cardiomyocytes; as a result, attempts to track PKD1 mobility shifts that result from agonist activation were only moderately successful. In contrast, PKD2 and PKD3 are recovered from resting cardiac fibroblasts and cardiomyocytes as single molecular species; both enzymes display robust mobility shifts in Phos-tag SDS-PAGE in response to treatment with sphingosine-1-phosphate, thrombin, PDGF, or H 2 O 2 . Studies with GF109203X implicate protein kinase C activity in the stimulus-dependent pathways that activate PKD2/PKD3 in both cardiac fibroblasts and cardiomyocytes. Studies with C3 toxin identify a novel role for Rho in the sphingosine-1-phosphate and thrombin receptor-dependent pathways that lead to the phosphorylation of PKD2/3 and the downstream substrate CREB in cardiomyocytes. In conclusion, Phos-tag SDS-PAGE provides a general screen for stimulus-specific changes in PKD2 and PKD3 phosphorylation and exposes a novel role for these enzymes in specific stress-dependent pathways that influence cardiac remodeling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35: , 1727-1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr(50) and Ser(645) (in resting and PMA-treated cardiomyocytes) as well as Thr(37), Thr(38), Ser(130), Thr(164), Thr(211), Thr(215), Ser(218), Thr(295), Ser(299) and Thr(656) (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser(130) and Thr(141) (sites just N-terminal to the pseudosubstrate domain). We show that S130D and T141E substitutions co-operate to increase PKCδ's basal lipid-independent activity and that Ser(130)/Thr(141) di-phosphorylation influences PKCδ's substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser(357), an ATP-positioning G-loop site that limits PKCδ's threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35: , 1727-1740]. The present study shows that S130D and T141E substitutions increase PKCδ's threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser(357). A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKCδ's maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser(130)/Thr(141) phosphorylations relieve auto-inhibitory constraints that limit PKCδ's activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity., (© 2016 Authors; published by Portland Press Limited.)

Protein kinase C (PKC) is comprised of a family of signal-regulated enzymes that play pleiotropic roles in the control of many physiological and pathological responses. PKC isoforms are traditionally viewed as allosterically activated enzymes that are recruited to membranes by growth factor receptor-generated lipid cofactors. An inherent assumption of this conventional model of PKC isoform activation is that PKCs act exclusively at membrane-delimited substrates and that PKC catalytic activity is an inherent property of each enzyme that is not altered by the activation process. This traditional model of PKC activation does not adequately explain the many well-documented actions of PKC enzymes in mitochondrial, nuclear, and cardiac sarcomeric (non-sarcolemmal) subcellular compartments. Recent studies address this dilemma by identifying stimulus-specific differences in the mechanisms for PKC isoform activation during growth factor activation versus oxidative stress. This review discusses a number of non-canonical redox-triggered mechanisms that can alter the catalytic properties and subcellular compartmentation patterns of PKC enzymes. While some redox-activated mechanisms act at structural determinants that are common to all PKCs, the redox-dependent mechanism for PKCδ activation requires Src-dependent tyrosine phosphorylation of a unique phosphorylation motif on this enzyme and is isoform specific. Since oxidative stress contributes to pathogenesis of a wide range of clinical disorders, these stimulus-specific differences in the controls and consequences of PKC activation have important implications for the design and evaluation of PKC-targeted therapeutics.

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr(313)-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr(313)-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser(359). We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser(359.) In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser(359) phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr(313) docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Protein kinase D1 (PKD1) is a Ser/Thr kinase implicated in a wide variety of cellular responses. PKD1 activation is generally attributed to a PKC-dependent pathway that leads to phosphorylation of the activation loop at Ser(744)/Ser(748). This modification increases catalytic activity, including that toward an autophosphorylation site (Ser(916)) in a postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding motif at the extreme COOH terminus. However, there is growing evidence that PKD1 activation can also result from a PKC-independent autocatalytic reaction at Ser(744)/Ser(748) and that certain stimuli increase in PKD1 phosphorylation at Ser(744)/S(748) without an increase in autophosphorylation at Ser(916). This study exposes a mechanism that results in a discrepancy between PKD1 COOH-terminal autocatalytic activity and activity toward other substrates. We show that PKD1 constructs harboring COOH-terminal epitope tags display high levels of in vitro activation loop autocatalytic activity and activity toward syntide-2 (a peptide substrate), but no Ser(916) autocatalytic activity. Cell-based studies show that the COOH-terminal tag, adjacent to PKD1's PDZ1-binding motif, does not grossly influence PKD1 partitioning between soluble and particulate fractions in resting cells or PKD1 translocation to the particulate fraction following treatment with PMA. However, a COOH-terminal tag that confers a high level of activation loop autocatalytic activity decreases the PKC requirement for agonist-dependent PKD1 activation in cells. The recognition that COOH-terminal tags alter PKD1's pharmacological profile is important from a technical standpoint. The altered dynamics and activation mechanisms for COOH-terminal-tagged PKD1 enzymes also could model the signaling properties of localized pools of enzyme anchored through the COOH terminus to PDZ domain-containing scaffolding proteins., (Copyright © 2014 the American Physiological Society.)

Two functionally important β1-adrenergic receptor (β1AR) polymorphisms have been identified. The R389G polymorphism influences coupling to the Gs-cAMP pathway. R(389)-β1ARs display enhanced activation of cAMP/PKA; they provide short-term inotropic support but also cause a predisposition to cardiomyopathic decompensation. A second S49G polymorphism is implicated in the evolution of heart failure, but the mechanism remains uncertain. This study shows that position 49 and 389 polymorphisms function in a coordinate manner to influence agonist-dependent cAMP/PKA and ERK responses. cAMP/PKA and ERK responses are more robust in HEK293 cells that heterologously overexpress G(49)-β1ARs, compared with S(49)-β1ARs. However, this phenotype is most obvious on a G(389)-β1AR background; the more robust agonist-dependent cAMP/PKA and ERK responses in R(389)-β1AR cells effectively obscure the effect of the S49G polymorphism. We also show that isoproterenol (Iso) and carvedilol activate ERK via a similar EGFR-independent mechanism in cells expressing various β1AR haplotypes. However, Iso activates ERK via an Src-independent pathway, but carvedilol-dependent ERK activation requires Src. Since the S49G polymorphism has been linked to changes in β1AR trafficking, we examined whether β1AR polymorphisms influence partitioning to lipid raft membranes. Biochemical fractionation studies show that all four β1AR variants are recovered in buoyant flotillin-enriched membranes; the distinct signaling phenotypes of the different β1AR variants could not be attributed to any gross differences in basal compartmentalization to lipid raft membranes. The allele-specific differences in β1AR signaling phenotypes identified in this study could underlie interindividual differences in responsiveness to β-blocker therapy and clinical outcome in heart failure.

Oxidative stress accompanies a wide spectrum of clinically important cardiac disorders, including ischemia/reperfusion, diabetes mellitus, and hypertensive heart disease. Although reactive oxygen species (ROS) can activate signaling pathways that contribute to ischemic preconditioning and cardioprotection, high levels of ROS induce structural modifications of the sarcomere that impact on pump function and the pathogenesis of heart failure. However, the precise nature of the redox-dependent change in contractility is determined by the source/identity of the oxidant species, the level of oxidative stress, and the chemistry/position of oxidant-induced posttranslational modifications on individual proteins within the sarcomere. This review focuses on various ROS-induced posttranslational modifications of myofilament proteins (including direct oxidative modifications of myofilament proteins, myofilament protein phosphorylation by ROS-activated signaling enzymes, and myofilament protein cleavage by ROS-activated proteases) that have been implicated in the control of cardiac contractility.

The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.

Protein kinase C (PKC) isoforms have emerged as important regulators of cardiac contraction, hypertrophy, and signaling pathways that influence ischemic/reperfusion injury. This review focuses on newer concepts regarding PKC isoform-specific activation mechanisms and actions that have implications for the development of PKC-targeted therapeutics.

Protein kinase D1 (PKD1) is a stress-activated serine/threonine kinase that plays a vital role in various physiologically important biological processes, including cell growth, apoptosis, adhesion, motility, and angiogenesis. Dysregulated PKD1 expression also contributes to the pathogenesis of certain cancers and cardiovascular disorders. Studies to date have focused primarily on the canonical membrane-delimited pathway for PKD1 activation by G protein-coupled receptors or peptide growth factors. Here, agonist-dependent increases in diacylglycerol accumulation lead to the activation of protein kinase C (PKC) and PKC-dependent phosphorylation of PKD1 at two highly conserved serine residues in the activation loop; this modification increases PKD1 catalytic activity, as assessed by PKD1 autophosphorylation at a consensus phosphorylation motif at the extreme C terminus. However, recent studies expose additional controls and consequences for PKD1 activation loop and C-terminal phosphorylation as well as additional autophosphorylation reactions and trans-phosphorylations (by PKC and other cellular enzymes) that contribute to the spatiotemporal control of PKD1 signaling in cells. This review focuses on the multisite phosphorylations that are known or predicted to influence PKD1 catalytic activity and may also influence docking interactions with cellular scaffolds and trafficking to signaling microdomains in various subcellular compartments. These modifications represent novel targets for the development of PKD1-directed pharmaceuticals for the treatment of cancers and cardiovascular disorders.

Oxidative stress is common in many clinically important cardiac disorders, including ischemia/reperfusion, diabetes, and hypertensive heart disease. Oxidative stress leads to derangements in pump function due to changes in the expression or function of proteins that regulate intracellular Ca(2+) homeostasis. There is growing evidence that the cardiodepressant actions of reactive oxygen species (ROS) also are attributable to ROS-dependent signaling events in the sarcomere. This minireview focuses on myofilament protein post-translational modifications induced by ROS or ROS-activated signaling enzymes that regulate cardiac contractility.

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of β-adrenergic receptor (β-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced β-AR insensitivity and the accompanying reduction in cell surface β-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in β-AR responsiveness. This can be attributed to PKC activation and reduced β-AR levels.

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α(1)-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α(1)-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser(133) and cTnI-Ser(23)/Ser(24) phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser(133) phosphorylation; there is no associated increase in cTnI-Ser(23)/Ser(24) phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser(133) phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.

Protein kinase C-δ (PKCδ) exerts important cardiac actions as a lipid-regulated kinase. There is limited evidence that PKCδ also might exert an additional kinase-independent action as a regulator of the subcellular compartmentalization of binding partners such as Shc (Src homologous and collagen), a family of adapter proteins that play key roles in growth regulation and oxidative stress responses. This study shows that native PKCδ forms complexes with endogenous Shc proteins in H(2)O(2)-treated cardiomyocytes; H(2)O(2) treatment also leads to the accumulation of PKCδ and Shc in a detergent-insoluble cytoskeletal fraction and in mitochondria. H(2)O(2)-dependent recruitment of Shc isoforms to cytoskeletal and mitochondrial fractions is amplified by wild-type-PKCδ overexpression, consistent with the notion that PKCδ acts as a signal-regulated scaffold to anchor Shc in specific subcellular compartments. However, overexpression studies with kinase-dead (KD)-PKCδ-K376R (an ATP-binding mutant of PKCδ that lacks catalytic activity) are less informative, since KD-PKCδ-K376R aberrantly localizes as a constitutively tyrosine-phosphorylated enzyme to detergent-insoluble and mitochondrial fractions of resting cardiomyocytes; relatively little KD-PKCδ-K376R remains in the cytosolic fraction. The aberrant localization and tyrosine phosphorylation patterns for KD-PKCδ-K376R do not phenocopy the properties of native PKCδ, even in cells chronically treated with GF109203X to inhibit PKCδ activity. Hence, while KD-PKCδ-K376R overexpression increases Shc localization to the detergent-insoluble and mitochondrial fractions, the significance of these results is uncertain. Our studies suggest that experiments using KD-PKCδ-K376R overexpression as a strategy to competitively inhibit the kinase-dependent actions of native PKCδ or to expose the kinase-independent scaffolding functions of PKCδ should be interpreted with caution.

Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

Protein kinase C-delta (PKCdelta) is a Ser/Thr kinase that regulates a wide range of cellular responses. This study identifies novel in vitro PKCdelta autophosphorylation sites at Thr(141) adjacent to the pseudosubstrate domain, Thr(218) in the C1A-C1B interdomain, Ser(295), Ser(302), and Ser(304) in the hinge region, and Ser(503) adjacent to Thr(505) in the activation loop. Cell-based studies show that Thr(141) and Thr(295) also are phosphorylated in vivo and that Thr(141) phosphorylation regulates the kinetics of PKCdelta downregulation in COS7 cells. In vitro studies implicate Thr(141) and Thr(295) autophosphorylation as modifications that regulate PKCdelta activity. A T141D substitution markedly increases basal lipid-independent PKCdelta activity; the PKCdelta-T141D mutant is only slightly further stimulated in vitro by PMA treatment, suggesting that Thr(141) phosphorylation relieves autoinhibitory constraints that limit PKCdelta activity. Mutagenesis studies also indicate that a phosphorylation at Thr(295) contributes to the control of PKCdelta substrate specificity. We previously demonstrated that PKCdelta phosphorylates the myofilament protein cardiac troponin I (cTnI) at Ser(23)/Ser(24) when it is allosterically activated by lipid cofactors and that the Thr(505)/Tyr(311)-phosphorylated form of PKCdelta (that is present in assays with Src) acquires as additional activity toward cTnI-Thr(144). Studies reported herein show that a T505A substitution reduces PKCdelta-Thr(295) autophosphorylation and that a T295A substitution leads to a defect in Src-dependent PKCdelta-Tyr(311) phosphorylation and PKCdelta-dependent cTnI-Thr(144) phosphorylation. These results implicate PKCdelta-Thr(295) autophosphorylation as a lipid-dependent modification that links PKCdelta-Thr(505) phosphorylation to Src-dependent regulation of PKCdelta catalytic function. Collectively, these studies identify novel regulatory autophosphorylations on PKCdelta that serve as markers and regulators of PKCdelta activity.

p66Shc is an adapter protein that is induced by hypertrophic stimuli and has been implicated as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. This study implicates p66Shc in an alpha(1)-adrenergtic receptor (alpha(1)-AR) pathway that requires the cooperative effects of protein kinase (PK)Cepsilon and PKCdelta and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. alpha(1)-ARs promote p66Shc-YY(239/240) phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCepsilon activity. alpha(1)-ARs also increase p66Shc-S(36) phosphorylation via an EGFR transactivation pathway involving PKCdelta. p66Shc links alpha(1)-ARs to an AKT signaling pathway that selectively phosphorylates/inactivates FOXO transcription factors and downregulates the ROS-scavenging protein manganese superoxide dismutase (MnSOD); the alpha(1)-AR-p66Shc-dependent pathway involving AKT does not regulate GSK3. Additional studies show that RNA interference-mediated downregulation of endogenous p66Shc leads to the derepression of FOXO3a-regulated genes such as MnSOD, p27Kip1, and BIM-1. p66Shc downregulation also increases proliferating cell nuclear antigen expression and induces cardiomyocyte hypertrophy, suggesting that p66Shc exerts an antihypertrophic action in neonatal cardiomyocytes. The novel alpha(1)-AR- and ROS-dependent pathway involving p66Shc identified in this study is likely to contribute to cardiomyocyte remodeling and the evolution of heart failure.

Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2DeltaCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin's effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin.

Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser744 and Ser748 followed by PKD1 autophosphorylation at Ser916. Although PKD-Ser916 autophosphorylation is widely used to track cellular PKD activity, this study exposes conditions leading to increased PKD-Ser(P)916 immunoreactivity without an associated increase in PKD activity in cardiomyocytes that heterologously overexpress catalytically inactive PKD1 and in cardiomyocytes treated with Gö6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser916-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser916 autophosphorylation can proceed via either an intermolecular reaction or an intramolecular autophosphorylation that requires only very low ATP concentrations that do not support target substrate phosphorylation. Additional studies show that Ser744 and Ser748 are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser916-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser744. However, the S916A substitution leads to a Ser748 phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser744 phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser916 autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser916 is required for subsequent autophosphorylation at Ser748. Finally, this study exposes a novel role for Ser916 and/or Ser748 autophosphorylation to terminate the cellular PKD1 signaling response.

Phosphorylation of cardiac troponin is a key mechanism involved in regulation of contractile function. In vitro kinase assays revealed that lysates prepared from resting cardiomyocytes contain cardiac troponin I (cTnI) and cTnT kinase activity. cTnI phosphorylation is inhibited by pharmacologic inhibitors of PKA, PKC, Rho kinase and PKC effectors such as RSK and PKD; these kinase inhibitors do not inhibit phosphorylation of cTnT. Rather, cTnT phosphorylation is decreased by the Raf inhibitor GW5074. In vitro kinase assays show that recombinant Raf phosphorylates cTnT, and that Raf-dependent cTnT phosphorylation is abrogated by a T206E substitution; Raf does not phosphorylate cTnI. These studies identify Raf-dependent cTnT-Thr(206) phosphorylation as a novel mechanism that would link growth factor-dependent signaling pathways to dynamic changes in cardiac contractile function.

Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCdelta to caveolae and increase PKCdelta-Tyr(311) phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Many growth regulatory stimuli promote cAMP response element-binding protein (CREB) Ser(133) phosphorylation, but the physiologically relevant CREB-Ser(133) kinase(s) in the heart remains uncertain. This study identifies a novel role for protein kinase D (PKD) as an in vivo cardiac CREB-Ser(133) kinase. We show that thrombin activates a PKCdelta-PKD pathway leading to CREB-Ser(133) phosphorylation in cardiomyocytes and cardiac fibroblasts. alpha(1)-Adrenergic receptors also activate a PKCdelta-PKD-CREB-Ser(133) phosphorylation pathway in cardiomyocytes. Of note, while the epidermal growth factor (EGF) promotes CREB-Ser(133) phosphorylation via an ERK-RSK pathway in cardiac fibroblasts, the thrombin-dependent EGFR transactivation pathway leading to ERK-RSK activation does not lead to CREB-Ser(133) phosphorylation in this cell type. Adenoviral-mediated overexpression of PKCdelta (but not PKCepsilon or PKCalpha) activates PKD; PKCdelta and PKD1-S744E/S748E overexpression both promote CREB-Ser(133) phosphorylation. Pasteuralla multocida toxin (PMT), a direct Galpha(q) agonist that induces robust cardiomyocyte hypertrophy, also activates the PKD-CREB-Ser(133) phosphorylation pathway, leading to the accumulation of active PKD and Ser(133)-phosphorylated CREB in the nucleus, activation of a CRE-responsive promoter, and increased Bcl-2 (CREB target gene) expression in cardiomyocyte cultures. Cardiac-specific Galpha(q) overexpression also leads to an increase in PKD-Ser(744)/Ser(748) and CREB-Ser(133) phosphorylation as well as increased Bcl-2 protein expression in the hearts of transgenic mice. Collectively, these studies identify a novel Galpha(q)-PKCdelta-PKD-CREB-Ser(133) phosphorylation pathway that is predicted to contribute to cardiac remodeling and could be targeted for therapeutic advantage in the setting of heart failure phenotypes.

Heterotrimeric G proteins are comprised of a guanine nucleotide binding Galpha subunit and the Gbetagamma dimers that link G protein-coupled receptors (GPCRs) to effectors. This study focuses on the expression and localization patterns for certain Gbeta and Ggamma subunits in neonatal and adult cardiomyocytes. We identify developmental downregulation of Gbeta1, Gbeta2, and Ggamma2, and a switch in the molecular form of Ggamma3, in cardiomyocytes. Gbeta1 is highly localized to caveolae membranes, whereas Gbeta2 is identified in caveolae and other membrane fractions. Gbeta3 is not detected in neonatal cardiomyocytes, but rather Gbeta3 is upregulated in adult cardiomyocytes and detected in the caveolae and soluble fractions. The observation that cardiomyocytes co-express multiple Gbeta and Ggamma subunits in a developmentally regulated manner, and that these Gbeta and Ggamma subunits assume distinct subcellular localization patterns, provides for a high level of signaling specificity in the heart.

Neutrophils are thought to orchestrate myocardial remodeling during the early progression to cardiac failure through the release of reactive oxygen species, antimicrobial peptides, and proteases. Although neutrophil activation may be beneficial at early stages of disease, excessive neutrophil infiltration can induce cardiomyocyte death and tissue damage. The neutrophil-derived serine protease cathepsin G (Cat.G) has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis. However, the involved signaling mechanisms for Cat.G are not well understood. This study identifies epidermal growth factor receptor (EGFR) transactivation as a mechanism whereby Cat.G induces signaling in cardiomyocytes. Cat.G induced a rapid and transient increase in EGFR tyrosine phosphorylation, and inhibition of EGFR kinase activity, either with AG1478 or by expression of kinase inactive EGFR mutants (EGFR-CD533), markedly attenuated EGFR downstream signaling and myocyte anoikis induced by Cat.G. Consistent with this effect of EGFR, high level expression of wild-type EGFR was sufficient to promote myocyte apoptosis. We also found that matrix metalloproteinase-dependent membrane shedding of heparin-binding EGF was involved in Cat.G signaling and that membrane type 1 matrix metalloproteinase activation may constitute a potential target that entails matrix metalloproteinase activation induced by Cat.G. The paradoxical proapoptotic effect of EGFR appeared to be dependent on protein tyrosine phosphatase SHP2 (Src homology domain 2-containing tyrosine phosphatase 2) activation and focal adhesion kinase downregulation. These results show that Cat.G-induced cardiomyocyte apoptosis involves an increase in EGFR-dependent activation of SHP2 that promotes focal adhesion kinase dephosphorylation and subsequent cardiomyocyte anoikis.

Post-translational modification, such as protein phosphorylation, plays a critical role to reversibly amplify and modulate signaling pathways. Since kinases and phosphatases have broad substrate recognition motifs, compartmentalization and localization of signaling complexes are required to achieve specific signals. Scaffolds are proteins that associate with two or more binding partners and function to enhance the efficiency and/or specificity of cellular signaling pathways. The identification of scaffolding proteins that control the tempo and/or spatial organization of signaling pathways in cells has benefited enormously from recent technological advances that allow for the detection of protein-protein interactions, including in vivo in intact cells. This review will focus on scaffolding proteins that nucleate multi-protein complexes (and could represent novel entry points into signaling pathways that might be amenable to therapeutic manipulation) in cardiomyocytes.

Protein kinase Cdelta (PKCdelta) is unusual among AGC kinases in that it does not require activation loop (Thr(505)) phosphorylation for catalytic competence. Nevertheless, Thr(505) phosphorylation has been implicated as a mechanism that influences PKCdelta activity. This study examines the controls of PKCdelta-Thr(505) phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCdelta autophosphorylation in the "priming" maturational PKCdelta-Thr(505) phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCdelta-Thr(505) phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the alpha(1)-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCepsilon overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCdelta-Thr(505) autophosphorylation and PKCdelta-Thr(505) phosphorylation in trans by PKCepsilon. Our studies show that adenovirus-mediated PKCepsilon overexpression leads to an increase in PKCdelta-Thr(505) phosphorylation. However, this cannot be attributed to an effect of PKCepsilon to function as a direct PKCdelta-Thr(505) kinase, since the PKCepsilon-dependent increase in PKCdelta-Thr(505) phosphorylation is accompanied by (and dependent upon) increased PKCdelta phosphorylation at Tyr(311) and Tyr(332). Further studies implicate Src in this mechanism, showing that 1) PKCepsilon overexpression increases PKCdelta-Thr(505) phosphorylation in cardiomyocytes and Src(+) cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCdelta-Tyr(311)/Tyr(332) phosphorylation), and 2) in vitro PKCdelta-Thr(505) autophosphorylation is augmented in assays performed with Src (which promotes PKCdelta-Tyr(311)/Tyr(332) phosphorylation). Collectively, these results identify a novel PKCdelta-Thr(505) autophosphorylation mechanism that is triggered by PKCepsilon overexpression and involves Src-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation.

AKT is a potent antiapoptotic kinase, but its role in the cardioprotective actions of alpha(1)-adrenergic receptors (ARs) remains uncertain, because alpha(1)-ARs typically induce little-to-no AKT activation in most cardiomyocyte models. This study identifies a prominent alpha(1)-AR-dependent AKT activation pathway that is under tonic inhibitory control by novel protein kinase Cs (nPKCs) in neonatal rat cardiomyocyte cultures. We also implicate Pyk2, Pyk2 complex formation with PDK-1 and paxillin, and increased PDK-1-Y373/376 phosphorylation as the mechanism that links alpha(1)-AR activation to increased AKT phosphorylation. nPKCs (which are prominent alpha(1)-AR effectors) interfere with this alpha(1)-AR-dependent AKT activation by blocking Pyk2/PDK-1/paxillin complex formation and PDK-1-Y373/376 phosphorylation. Additional studies used an adenoviral-mediated overexpression strategy to show that Pyk2 exerts dual controls on antiapoptotic PDK-1/AKT and proapoptotic c-Jun N-terminal kinase (JNK) pathways. Although the high nPKC activity of most cardiomyocyte models favors Pyk2 signaling to JNK (and cardiac apoptosis), the cardioprotective actions of Pyk2 through the PDK-1/AKT pathway are exposed when PKC or JNK activation is prevented. Collectively, these studies identify JNK and AKT as functionally distinct downstream components of the alpha(1)-AR/Pyk2 signaling pathway. We also implicate nPKCs as molecular switches that control the balance of signaling via proapoptotic JNK and antiapoptotic PDK-1/AKT pathways, exposing a novel mechanism for nPKC-dependent regulation of cardiac hypertrophy and failure.

Thrombin activates protease-activated receptor-1 (PAR-1) and engages signaling pathways that influence the growth and survival of cardiomyocytes as well as extracellular matrix remodeling by cardiac fibroblasts. This study examines the role of Shc proteins in PAR-1-dependent signaling pathways that influence ventricular remodeling. We show that thrombin increases p46Shc/p52Shc phosphorylation at Tyr(239)/Tyr(240) and Tyr(317) (and p66Shc-Ser(36) phosphorylation) via a pertussis toxin-insensitive epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts; p66Shc-Ser(36) phosphorylation is via a MEK-dependent mechanism. In contrast, cardiac fibroblasts express beta(2)-adrenergic receptors that activate ERK through a pertussis toxin-sensitive EGFR transactivation pathway that does not involve Shc isoforms or lead to p66Shc-Ser(36) phosphorylation. In cardiomyocytes, thrombin triggers MEK-dependent p66Shc-Ser(36) phosphorylation, but this is not via EGFR transactivation (or associated with Shc-Tyr(239)/Tyr(240) and/or Tyr(317) phosphorylation). Importantly, p66Shc protein expression is detected in neonatal, but not adult, cardiomyocytes; p66Shc expression is induced (via a mechanism that requires protein kinase C and MEK activity) by Pasteurella multocida toxin, a Galpha(q) agonist that promotes cardiomyocyte hypertrophy. These results identify novel regulation of individual Shc isoforms in receptor-dependent pathways leading to cardiac hypertrophy and the transition to heart failure. The observations that p66Shc expression is induced by a Galpha(q) agonist and that PAR-1 activation leads to p66Shc-Ser(36) phosphorylation identifies p66Shc as a novel candidate hypertrophy-induced mediator of cardiomyocyte apoptosis and heart failure.